A third generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airways of 11 volunteers with cyctic fibrosis. Assessments of does limiting toxicity (DLT), efficiency of gene transfer and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flu-like symptoms and transient radiographic infiltrates, was seen at 2.1 x 10-11 total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization of an oligoprobe against unique vector sequence Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation. Expression of transgene did not appear to be related to the dose of vector administered and was no longer detectable after 43 days. Specific cell-mediated immunity was induced in most subjects but was not does related. However, airway administration of recombinant adenovirus resulted in only a mild systemic humoral immune response in most subjects. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors that efficiency is low and of short duration in the native CF airway. We hypothesize that gene transfer will be more evenly distributed and will be associated with less toxivity at high doses using an aerosol delivery system than with the previous liquid drip technique. We therefore propose to modify the current study using aerosol delivery of the third generation adenoviral vector to achieve a more uniform delivery of vector with less alvelor deposition of virus. We predict than an aerosol preparation will allow one to administer a higher dose of vector with less intrapulmonary inflammation and better airway distribution and gene transfer.